The overall objective of this project is to elucidate the precise molecular mechanisms responsible for force maintenance in vascular smooth muscle (VSM). A primary event in the initiation of smooth muscle contraction is Ca2+-calmodulin dependent phosphorylation of the 20 kd myosin light chains (MLC). Although MLC phosphorylation has been shown to be an important regulator of smooth muscle contraction, an increasing body of literature has clearly demonstrated that another regulatory system for force exists, and this second system maybe even more important for force maintenance. The purpose of this proposed research project is to test the hypothesis that protein kinase C (PKC) nad caldesmon (CDM) are directly involved in this second regulatory system for force. In order to test this hypothesis, the specific aims are: 1) To determine the calcium sensitivity of force produced by activation of PKC; 2) To determine the role of activation of PKC for force developed during both the warming and alpha-agonists stimulated contractions; 3) To determine the effects of inhibiting PKC function on the calcium sensitivity of force; and 4) To determine the effects of inhibiting CDM function on the calcium sensitivity of force. In order to fulfill these specific aims, experiments will be performed with single, freshly dissociated VSM cells. Using a technique I have developed to isolate contractile single VSM cells from the aorta and attach these cells to microtools, force can be recorded. After detergent skinning, force is recorded both during activation (increasing [Ca2+]) and relaxation (decreasing [Ca2+]). To test the roles of PKC and CDM in force maintenance, force will be measured in solutions containing selective inhibitors of PKC and CDM function. In addition to directly probe the mechanism for pharmacomechanical coupling, force will be measured during alpha-agonist stimulated contractions in alpha-toxin skinned single VSM cells microinjected with inhibitors of PKC and/or CDM function. if PKC and/or CDM are involved in the mechanism of force maintenance, one would predict that inhibition of their function would result in relaxation at activating [Ca2+]. Experiments measuring MLC phosphorylation produced by activation of PKC and/or CDM. The information gained from this proposal will provide direct evidence for the role(s) of PKC and/or CDM in the regulation of force in smooth muscle contraction.